Synthesis of Tridecaptin–Antibiotic Conjugates ... - ACS Publications

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Synthesis of Tridecaptin−Antibiotic Conjugates with in Vivo Activity against Gram-Negative Bacteria Stephen A. Cochrane,† Xuefeng Li,‡ Sisi He,‡ Min Yu,‡ Min Wu,‡ and John C. Vederas*,† †

Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada Department of Biomedical Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203-9037, United States

‡

S Supporting Information *

ABSTRACT: A series of tridecaptin−antibiotic conjugates were synthesized and evaluated for in vitro and in vivo activity against Gram-negative bacteria. Covalently linking unacylated tridecaptin A1 (H-TriA1) to rifampicin, vancomycin, and erythromycin enhanced their activity in vitro but not by the same magnitude as coadministration of the peptide and these antibiotics. The antimicrobial activities of the conjugates were retained in vivo, with the H-TriA1-erythromycin conjugate proving a more effective treatment of Klebseilla pneumoniae infections in mice than erythromycin alone or in combination with H-TriA1.

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INTRODUCTION The emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) bacteria is a major concern worldwide. A recent U.K. report predicts that by 2050, antibiotic resistance will have caused 300 million premature deaths and cost the global economy $100 trillion.1 Of particular concern are Gramnegative bacteria. In the past 40 years, only four new classes of structurally and mechanistically distinct antibiotics have gained clinical approval for the treatment of systemic infections (daptomycin, linezolid, fidaxomicin, and bedaquiline); however, none of these are active against Gram-negative bacteria.2,3 Currently there are two new classes of Gram-negative targeting antibiotics in the clinical pipeline that are, or are modeled on, antimicrobial peptides (macrocyclic peptide POL7080 and peptidomimetic brilacidin).4,5 Antimicrobial peptides are playing an increasingly large role in targeting MDR bacteria.6 Given that many antimicrobial peptides target the bacterial membrane and it is difficult for bacteria to reorganize their membrane, resistance development is often limited.7 Therefore, it is not surprising that new classes of antibiotics that are based on peptides are being actively investigated. Over the past several years our group has been working on a class of antimicrobial lipopeptides known as the tridecaptins. These nonribosomal peptide synthetase products are Nterminally acylated tridecapeptides (Figure 1) that display © XXXX American Chemical Society

Figure 1. Synthetic and natural analogues of tridecaptin A1. Received: October 9, 2015

A

DOI: 10.1021/acs.jmedchem.5b01578 J. Med. Chem. XXXX, XXX, XXX−XXX

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strong activity against Gram-negative bacteria while showing low toxicity toward mammalian cells.8−11 Octyltridecaptin A1 (Oct-TriA1) (1), a synthetic analogue of tridecaptin A1 (2), has low μM activity against MDR Klebsiella pneumoniae and other Gram-negative organisms. H-TriA1 (3) is an unacylated analogue of tridecaptin A1 that is substantially less active than the natural peptide and has good serum stability.12 However, H-TriA1 is a sensitizer of the outer membrane (OM) of Gramnegative bacteria and coadministrations of subminimum inhibitory concentrations (MIC) of this peptide with antibiotics like rifampicin and vancomycin dramatically increase their activity.12 An emerging strategy in combination therapy is the use of heterodimer antibiotics, in which two different antibiotics are covalently linked together.13 Some of the potential advantages of this approach over individual antibiotics are renewed activity against MDR bacteria, enhanced efficacy and duration in vivo, expanded spectrum of activity, and reduced development of resistance.14 We postulated that covalently linking tridecaptin A1 to antibiotics typically used in the treatment of Gram-positive infections would facilitate their passage across the OM of Gram-negative bacteria. This Trojan horse approach has been previously performed through covalent linkage of antibiotics to siderophores,15,16 cell penetrating peptides,17,18 and polymers.19 This method could also offer some advantages over the coadministration of HTriA1 with antibiotics. If both H-TriA1 and the antibiotic do not reach the target bacteria in vivo, there will be no synergistic effect; however, conjugation will ensure the simultaneous delivery of the peptide and the antibiotic. Also, the moderate size of H-TriA1 and its stability to proteases may prevent efflux of the antibiotic from the bacterial cell and prevent it from being degraded by targeting enzymes. Therefore, we sought to investigate if tridecaptin−antibiotic conjugates would provide enhanced activity against Gram-negative bacteria.

linker was arbitrarily chosen for this purpose. The Fmocazidoamino acid 4 was first synthesized from commercially available Fmoc-Glu-OtBu (5). Acid 5 was coupled to H2NPEG3-N3 using HATU coupling conditions followed by tertbutyl ester deprotection with TFA to afford the Fmocazidoamino acid 4 in 45% yield (Scheme 1). Azido analogues Scheme 1. Synthesis of Fmoc-azidoamino Acid 4

of Oct-TriA1 and H-TriA1 (6 and 7, respectively) were then synthesized using Fmoc-SPPS (Figure 2). To ensure that the

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RESULTS AND DISCUSSION The first stage in designing a tridecaptin A1−antibiotic conjugate is identifying an appropriate linkage site on each compound. This must be in an area that is not involved in receptor binding in the compounds mechanism of action. Tridecaptin A1 has several possible linkage sites, including through the side chain amines of its Dab and D-Dab residues, the C-terminal carboxylate, and the side chain carboxylate of Glu10. A previous alanine scan of Oct-TriA1 has shown that while replacement of the Dab and D-Dab residues results in lower activity, Glu10 substitution has no effect.20 Also, as these peptides are synthesized by solid-phase peptide synthesis (SPPS), the C-terminal carboxylate is inaccessible for modification, as it is attached to a solid support. In contrast, Glu10 could be easily modified on-resin through orthogonal protection of the side chain carboxylate or by introduction of a premodified glutamic acid residue. Thus, we chose Glu10 as the conjugation site for modification of Oct-TriA1 and H-TriA1. Attachment of Oct-TriA1 to an antibiotic could provide enhanced antimicrobial activity, whereas H-TriA1 attachment would simply facilitate OM transport. Copper catalyzed azide alkyne cycloaddition (CuAAC) is a common strategy employed to link biomolecules due to its high tolerance to many different functional groups.21 Therefore, we decided to modify Glu10 to incorporate an azide moiety attached to a PEG chain. We rationalized that a linker would be required to prevent the tridecaptin analogue and antibiotic from disrupting binding to their respective targets. A PEG3

Figure 2. Structures of azidotridecaptin A1 analogs 6 and 7.

linker did not affect the respective antimicrobial or synergistic activities of 6 and 7, they were tested against E. coli ATCC 25922. Oct-TriA1 analogue 6 completely retained its antimicrobial activity (MIC = 3.13 μM), and coadministration of 12.5 μg/mL of 7 with rifampicin increased the activity of rifampicin 128-fold. With the azido peptides 6 and 7 in hand, we then proceeded to synthesize the complementary alkynyl antibiotics. We have previously shown that the antimicrobial activities of the hydrophobic antibiotics vancomycin and rifampicin are significantly increased by coadministration with sub-MIC concentrations of H-TriA1. This is due to disruption of the OM, making it more permeable to these antibiotics.12 Therefore, we selected vancomycin, rifampicin, and the macrolide erythromycin A, which has low activity against Gram-negative bacteria due to expulsion by efflux pumps, for conjugation to 6 and 7. Previous studies have shown that the carboxylic acid on vancomycin can be modified without loss of activity.22 Treatment of vancomycin (Van, 8) with propargylamine under HBTU coupling conditions yields fully active vancomycin alkyne (9) in good yield (Scheme 2).23 The ketone functionality on erythromycin A (Eryc) has previously been modified as an oxime ether and shown not to reduce activity.24,25 Treatment of commercially available erythromycin A oxime (10) with propargyl bromide yields erythromycin B

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Scheme 2. Synthesis of Antibiotics Containing an Alkyne Functional Group for Conjugation to the Azidotridecaptin Analogs

alkyne (11) in 30% yield (Scheme 2). Recent studies have shown that loss of the piperazine ring on rifampicin (Rif) results in a substantial decrease in activity;26 therefore we chose to replace the 4-methylpiperazine group with a 4-propargylpiperazine unit. 1-Amino-4-propargylpiperazine (12) was synthesized in three steps from piperazine (see Supporting Information). Addition of amine 12 to commercially available rifampicin aldehyde (13) yields rifampicin alkyne (14) in excellent yield. CuAAC was then used to link the azido peptides 6 and 7 to the antibiotic alkynes 9, 11, and 14, producing a total of six tridecaptin−antibiotic conjugates (15−20) in 29−59% yields (Scheme 3). These conjugates were tested against regular strains of the Gram-negative bacteria E. coli, K. pneumoniae, and A. baumannii and MDR strains of K. pneumoniae and A. baumannii (Table 1). For comparison, the antibiotics alone and in combination with 12.5 μg/mL of H-TriA1 were tested. In all instances, linking Oct-TriA1 to the antibiotics resulted in lower antimicrobial activity than the unmodified peptide. Linking HTriA1 to rifampicin also did not increase the in vitro activity of rifampicin. However, moderate activity increases were observed with H-TriA1-Van and H-TriA1-Eryc. Linking H-TriA1 to vancomycin resulted in a 16-fold increase in activity against E. coli, and an 8-fold increase was observed against MDR K.

Scheme 3. Synthesis of Tridecaptin−Antibiotic Conjugates

pneumoniae and A. baumannii. A lower increase in activity was found with H-TriA1-Eryc, which is 2- to 4-fold more active than erythromycin against most strains tested. Although conjugation of vancomycin or erythromycin to H-TriA1 increases their respective antimicrobial activities, these observed increases are lower than when they are mixed with a sub-MIC concentration of H-TriA1. For example, addition of H-TriA1 with rifampicin, vancomycin, or erythromycin results in respective 128-, 64-, and 8-fold increases in antimicrobial activity against E. coli. Given the moderate effort required to synthesize these heterodimers, coadministration with H-TriA1 is a much more attractive strategy. However, this synergistic effect may not C

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Table 1. In Vitro Activity of Tridecaptin−Antibiotic Conjugates against Gram-Negative Bacteriaa MIC (μM) compd

Ec

Kp

MDR Kp

Ab

MDR Ab

H-TriA1 Oct-TriA1 rifampicin Rif + H-TriA1 H-TriA1-Rif Oct-TriA1-Rif vancomycin Van + H-TriA1 H-TriA1-Van Oct-TriA1-Van erythromycin Eryc + H-TriA1 H-TriA1-Eryc Oct-TriA1-Eryc

100 3.13 6.25 0.05 25 3.13 200 1.56 25 12.5 100 3.13 50 12.5

100 3.13 12.5 0.005 50 6.25 200 0.78 100 100 100 0.4 50 3.13

100 6.25 25 0.1 100 100 250 25 200 25 400 200 200 12.5

200 6.25 0.4 0.05 25 6.25 200 3.13 25 25 200 12.5 50 6.25

200 6.25 0.78 0.05 50 6.25 200 200 25 50 200 200 25 6.25

a Ec = E. coli. Kp = K. pneumoniae. Ab = A. baumannii. In synergistic combinations, [H-TriA1] = 12.5 μg/mL. Regular strains = Ec ATCC 25922, Kp ATCC 13883, Ab ATCC 19606. MDR = Kp ATCC 700603, Ab ATCC BAA-1605.

persist in vivo, especially if the peptide and antibiotics do not arrive at the target organism together. Therefore, we further evaluated the activity of the tridecaptin−antibiotic conjugates in vivo. C57BL/6J mice were infected with K. pneumoniae (ATCC 43816) by intranasal instillation followed by injection (tail) 1 h later with the antibiotic alone (4 mg/kg), antibiotic (2 mg/kg) and H-TriA1 (2 mg/kg) or H-TriA1−antibiotic conjugate (4 mg/kg). The survival rates of mice treated with rifampicin (Figure 3A), erythromycin (Figure 3B), or vancomycin (Figure 3C) based treatments were monitored over 7 days. The remaining colony forming units per lung (CFU/lung) of moribund mice were also determined (Figure 4). In the negative control groups (DMSO), all mice infected with K. pneumoniae died within 3−5 days. Treatment with H-TriA1 moderately improved survival rates, with 40−50% of mice still alive after 7 days. Oct-TriA1 retained its activity in vivo, with an 80% survival rate at 4 mg/kg (not shown). Whereas rifampicin was an excellent treatment in vivo (100% survival), rifampicin + H-TriA1 and H-TriA1-Rif had lower survival rates. We were surprised to find that vancomycin treatment against K. pneumoniae was effective in vivo, given its poor in vitro activity; however the in vivo environment is much more complicated than an in vitro setting. For example, a secondary infection may occur in sick mice, which is susceptible to vancomycin. Also, the effective local concentration at the infection site may be sufficiently high to limit the infection. Addition of H-TriA1 with vancomycin led to survival rates of 60% and treatment with HTriA1-Van dropped further to 40%. Erythromycin alone or in combination with H-TriA1 was ineffective at treating the K. pneumoniae infection, with only 40% of mice surviving by day 7. However, the H-TriA1-Eryc conjugate increased the survival rate to 80%. These results were corroborated by determination of the CFU/lung (Figure 4). The lungs of moribund mice (which died during the survival experiment or were sacrificed at the end) were excised and homogenized, and the amount of Kp remaining in the lungs was determined. A lower CFU/lung count corresponds to a more effective treatment. This showed that H-TriA1-Eryc reduced the CFU/lung by approximately 3fold relative to the negative control. Therefore, linking

Figure 3. In vivo testing of compounds. Groups of mice (five) were infected with Kp followed by intravenous injection of the desired compound 1 h later. Each group was monitored for 1 week. The survival rates are shown for (A) rifampicin, (B) erythromycin, and (C) vancomycin based treatments.

erythromycin A to H-TriA1 improves its antimicrobial activity against K. pneumoniae. A similar reduction in the infection was observed with Oct-TriA1, confirming that the activity of the tridecaptins is retained in vivo.

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CONCLUSION The glutamic acid residue on tridecaptin A1 is a good site for synthetic modification without reducing antimicrobial activity. We have provided convenient one-step syntheses for alkyne analogues of the antibiotics vancomycin, rifampicin, and erythromycin and have successfully conjugated these into tridecaptin analogues using copper catalyzed azide alkyne cycloaddition. Linking unacylated tridecaptin A1 to erythromycin and vancomycin moderately improves their activity in vitro D

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cast) 3324, 3067, 2877, 2109, 1721 cm−1; 1H NMR (CDCl3, 500 MHz) δ 7.76 (d, 2H, J = 7.5 Hz, Fmoc-ArH), 7.60 (t, 2H, J = 7.0 Hz, Fmoc-ArH), 7.39 (t, 2H, J = 7.5 Hz, Fmoc-ArH), 7.31 (t, 2H, J = 7.4 Hz, Fmoc-ArH), 6.63−6.57 (m, 1H, OCH2CH2NH), 6.01 (d, 1H, J = 6.8 Hz, Fmoc-NH), 4.41−4.34 (m, 3H, Hα + Fmoc-CH2), 4.21 (t, 1H, J = 7.1 Hz, Fmoc-CH), 3.64−3.61 (m, 10H, 5 × OCH2), 3.56 (t, 2H, J = 4.9 Hz, OCH2CH2NH), 3.52−3.41 (m, 2H, OCH2CH2NH), 3.36 (t, 2H, J = 5.0 Hz, CH2N3), 2.51−2.37 (m, 2H, Hγ), 2.23−2.08 (m, 2H Hβ); HRMS (ES) calcd for C28H35N5O8Na [M + Na]+ 592.2378, found 592.2371. Peptide Synthesis. Solid-phase peptide synthesis was carried out on a 0.1 mmol scale using Fmoc chemistry on preloaded L-Ala-2chlorotrityl resin (ChemImpex, 0.824 mmol/g loading). Reactions were performed in a custom-built 20 mL glass fritted column fitted with a T-joint and three-way T-bore PTFE stopcock. The resin was preswollen by bubbling in DMF (5 mL, 10 min) with argon. Between deprotections and couplings the vessel was drained under argon pressure and washed with DMF (3 × 5 mL). The Fmoc group was removed by bubbling with 20% piperidine in DMF (3 × 5 mL × 5 min). The deprotection steps were monitored by UV (254 nm) detection of the washings. Fmoc-D-allo-Ile (5 equiv) was preactivated by shaking with HATU (5 equiv) and DIPEA (10 equiv) in DMF (5 mL) for 5 min. The resin was bubbled in the coupling solution for 1 h, drained, and washed with DMF (3 × 5 mL). The deprotection and coupling steps were continued to complete the peptide synthesis. The resin-bound peptide was washed with CH2Cl2 (3 × 5 mL) and dried under argon for 20 min. The resin was transferred to a screw top vial containing TFA/TIPS/H2O (95:2.5:2.5, 5 mL) and gently shaken for 2 h. The cleavage solution was filtered and concentrated in vacuo, and the crude peptide was precipitated with cold diethyl ether. The crude peptide was dissolved in H2O/MeCN (1:1, 5 mL) and purified using prep-scale HPLC: Phenomenex C18 column, flow rate 10 mL/min, detected at 220 nm. Gradient: Starting from 20% MeCN (0.1% TFA) and 80% water (0.1% TFA) for 5 min, ramping up to 55% MeCN over 30 min, then ramping up to 95% MeCN over 3 min, staying at 95% MeCN for 3 min, ramping down to 20% MeCN over 2 min, then staying at 20% MeCN for 5 min. The product containing fractions were pooled, concentrated, frozen, and lyophilized to yield the product as a white powder. Synthesis of Vancomycin Alkyne (9). Vancomycin·HCl·2H2O (115 mg, 75.6 μmol) was dissolved in dry DMSO (1 mL) and dry DMF (1 mL). Molecular sieves were added (320 mg, 4 Å), and the solution was left to stand at ambient temperature for 1 h. The molecular sieves were removed, and propargylamine·HCl (14 mg, 151.1 μmol) was added. The mixture was cooled to 0 °C, and HBTU (43 mg, 113.4 μmol) and DIPEA (80 μL, 435.5 μmol) were added. The mixture was warmed to ambient temperature and stirred overnight. The mixture was concentrated in vacuo, dissolved in 1:1 H2O/MeCN, and purified by HPLC: Gilson preparative system, Phenomenex C18 column, flow rate 10 mL/min, detected at 220 nm. Gradient: 20% MeCN (0.1% TFA) and 80% water (0.1% TFA) for 5 min, ramping up to 25% MeCN over 10 min, then ramping up to 95% MeCN over 1 min, staying at 95% MeCN for 2 min, ramping down to 20% MeCN over 1 min, then staying at 20% MeCN for 3 min. Product containing fractions were pooled, frozen, and lyophilized to yield the product as a white solid (76.5 mg, 68%). Retention time (analytical) = 3.3 min; 1H NMR (DMSO-d6, 500 MHz) δ 9.30 (s, 1H), 8.97−8.94 (m, 2H), 8.68 (m, 1H), 8.43 (d, 1H, J = 5.2 Hz), 8.34 (t, 1H, J = 5.5 Hz), 7.85 (d, 1H, J = 1.6 Hz), 7.60−7.44 (m, 5H), 7.33 (d, 1H, J = 8.3 Hz), 7.17−7.19 (m, 2H), 6.76−6.64 (m, 3H), 6.37 (d, 1H, J = 2.2 Hz), 6.23 (d, 1H, J = 2.2 Hz), 5.93 (br s, 1H), 5.84 (br s, 1H), 5.75 (d, 1H, J = 7.9 Hz), 5.61 (s, 1H), 5.44 (br s, 1H), 5.28−5.16 (m, 4H), 4.94− 4.90 (br s, 1H), 4.67 (app q, 1H, J = 6.4 Hz), 4.46−4.39 (m, 2H), 4.25−4.18 (m, 2H), 3.99−3.86 (m, 4H), 3.69−3.58 (m, 4H), 3.20− 3.10 (m, 4H), 2.65−2.60 (m, 2H), 2.15 (dd, 1H, J = 15.4, 7.6 Hz), 1.92−1.85 (m, 1H), 1.73−1.53 (m, 4H), 1.30−1.20 (m, 12H), 1.06 (d, 3H, J = 6.4 Hz), 0.91 (d, 3H, J = 6.2 Hz), 0.86 (d, 3H, J = 6.2 Hz); HRMS (ES) calcd for C69H78Cl2N10O23 [M + H]+ 1484.4618, found 1484.4618.

Figure 4. Amount of Kp remaining in lungs after treatment. Upon death, the lungs of all mice were excised and the CFU of Kp present in the lungs was determined.

but not to the same degree as coadministration of the peptide and these antibiotics. The antimicrobial activities of the tridecaptins and conjugates are retained in vivo. In particular, linking erythromycin to unacylated tridecaptin A1 was a more effective treatment of Klebsiella pneumoniae infections in mice than the antibiotic alone or by coadministration with the peptide. The previously reported octyltridecaptin A1 retains strong antimicrobial activity in vivo, highlighting the potential utility of the tridecaptins as Gram-negative targeting antibiotics.

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EXPERIMENTAL SECTION

General. NMR spectra were recorded on Varian Inova 500 and 600 spectrometers. For 1H NMR spectra, δ values were referenced to CDCl3 (7.26 ppm), DMSO-d6 (2.50 ppm), or D2O (4.79 ppm). For 13 C NMR spectra, δ values were referenced to CDCl3 (77.0 ppm). Mass spectra were recorded on an Aglient Technologies 6130 LCMS or an AB Sciex Voyager Elite MALDI with 4-hydroxy-α-cyanocinnamic acid as the matrix. Preparative HPLC was performed on a Gilson HPLC system equipped with a model 322 HPLC pump, GX-271 liquid handler, 156 UV/vis detector, and a 10 mL sample loop. Analytical HPLC was performed on a Gilson HPLC system equipped with a model 322 HPLC pump, FC 203B fraction collector, 171 diode array detector, and a Rheodyne 7225i injector fitted with a 1000 μL sample loop. The columns used were a Vydac C18 column (5 μm, 4.6 mm × 250 mm) for analytical scale and Phenomenex C18 column (5 μm, 21.2 mm × 250 mm) for preparative scale. Purity Analysis. All peptides, antibiotic alkynes, and conjugates were purified by preparative HPLC, and purity was then determined by analytical HPLC: Vydac C18 column, flow rate 1 mL/min, detected at 220 nm. Gradient: 20% MeCN (0.1% TFA) and 80% water (0.1% TFA) to 95% MeCN over 30 min, ramping down to 20% MeCN over 2 min and finishing at 20% MeCN for 2 min. In all cases products had >95% purity. Synthesis of Fmoc-Gln(PEG3N3)-OH (4). Fmoc-Glu-OtBu (1.00 g, 2.35 mmol) was dissolved in dry DMF (15 mL) and cooled to 0 °C. DIPEA (0.5 mL, 2.84 mmol) and HATU (1.08 g, 2.84 mmol) were then added. A solution of H2N-PEG3-N3 (0.56 mL, 2.84 mmol) in dry DMF (5 mL) was then added and the mixture stirred at ambient temperature for 18 h. The mixture was concentrated in vacuo and redissolved in EtOAc (100 mL). This solution was washed with 10% citric acid (50 mL), saturated NaHCO3 (50 mL), and brine (50 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The resulting oil was dissolved in CH2Cl2 (10 mL) and TFA (10 mL) and stirred at ambient temperature for 3 h. The reaction mixture was then concentrated in vacuo and purified by column chromatography (SiO2, EtOAc to 9:1 EtOAc/MeOH) to yield acid 4 as a colorless oil (0.73 g, 45% over 2 steps). [α]D25 6.03 (c 0.6 g/100 mL, EtOAc); IR (CHCl3 E

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Synthesis of Erythromycin Alkyne (11). To a solution of erythromycin A and 9-O-oxime (100 mg, 0.133 mmol) in MeCN (1.2 mL) were added propargyl bromide (80% solution in toluene, 18.4 μL, 0.165 mmol) and KOH (13 mg, 0.21 mmol). The mixture was stirred at ambient temperature for 3 h followed by the addition of CHCl3 (2 mL). The resulting mixture was filtered, concentrated in vacuo, and redissolved in 1:1 MeCN/H2O (3 mL). The product was purified by HPLC: Gilson preparative system, Phenomenex C18 column, flow rate 5 mL/min, detected at 220 nm. Gradient: starting from 10% MeCN (0.04% AcOH) and 90% water (0.04% AcOH), ramping up to 40% MeCN over 10 min, then ramping up to 95% MeCN over 1 min, staying at 95% MeCN for 2 min, ramping down to 20% MeCN over 1 min, then staying at 20% MeCN for 2 min. Product containing fractions were pooled, frozen, and lyophilized to yield the product as a white fluffy powder (31 mg, 30%). Retention time (analytical) = 12.1 min; 1H NMR (DMSO-d6, 500 MHz) δ 5.14 (dd, 1H, J = 11.1, 1.8 Hz), 4.75 (d, 1H, J = 4.8 Hz), 4.52−4.49 (m, 2H), 4.13 (br s, 1H), 4.06, (m, 1H) 4.00−3.95 (m, 1H), 3.84−3.76 (m, 2H), 3.65 (app td, 1H, J = 11.2, 3.8 Hz), 3.56−3.53 (m, 2H), 3.49 (d, 1H, J = 6.5 Hz), 3.24 (s, 3H), 3.15 (s, 3H), 3.09 (s, 3H), 2.93 (d, 1H, J = 9.4 Hz), 2.80 (app dt, 1H, J = 16.3, 7.2 Hz), 2.64 (app q, 1H, J = 7.0 Hz), 2.28 (d, 1H, J = 14.9 Hz), 2.07−2.02 (m, 1H), 1.99−1.94 (m, 1H), 1.86−1.78 (m, 1H), 1.65 (s, 1H), 1.60−1.48 (m, 3H), 1.41−1.23 (m, 5H), 1.19− 0.99 (m, 18H), 0.75 (t, 3H, J = 7.4 Hz); HRMS (ES) calcd for C40H70N2O13 [M + H]+ 786.4951, found 786.4941. Synthesis of Rifampicin Alkyne (14). Rifaldehyde (100 mg, 0.138 mmol) was suspended in dry THF (0.5 mL). 1-Amino-4propargylpiperazine (12) (19 mg, 0.138 mmol) was added and the mixture stirred vigorously for 15 min. The mixture was then diluted with CH2Cl2 (7 mL) and washed with 5.5 mL of a solution of ascorbic acid (2.0 g) in 3:1 H2O/brine (40 mL). The aqueous phase was then extracted with CH2Cl2 (7 mL) and the combined CH2Cl2 extracts were dried over anhydrous Na2SO4 and concentrated in vacuo to yield rifampicin alkyne (14) as a red solid (110 mg, 94%). Retention time (analytical) = 19.7 min; 1H NMR (CDCl3, 500 MHz) δ 12.02 (s, 1H), 8.29 (s, 1H), 6.58 (dd, 1H, J = 15.5, 12.3 Hz), 6.39 (d, 1H, J = 11.1 Hz), 6.21 (d, 1H, J = 13.6 Hz), 5.94 (dd, 1H J = 15.4, 5.0 Hz), 5.11 (dd, 1H, J = 12.7, 6.8 Hz), 4.95 (d, 1H, J = 10.7 Hz), 3.78−3.73 (m, 1H), 3.60 (d, 1H, J = 4.7 Hz), 3.48 (d, 1H, J = 6.8 Hz), 3.43 (s, 1H), 3.38 (s, 1H), 3.22−3.19 (m, 1H), 3.11−3.09 (m, 1H), 3.05−3.01 (m, 4H), 2.76−2.69 (m, 2H), 2.41−2.36 (m, 1H), 2.28 (s, 1H), 2.23 (s, 3H), 2.08−2.06 (m, 6H), 1.80 (s, 3H), 1.73−1.70 (m, 1H), 1.57−1.52 (m, 1H), 1.39−1.34 (m, 1H), 1.02 (d, 3H, J = 7.0 Hz), 0.90 (d, 3H, J = 7.1 Hz), 0.61 (d, 3H, J = 76.9 Hz), −0.30 (d, 3H, J = 6.9 Hz). HRMS (ES) calcd for C45H57N4O12 [M − H]− 845.3978, found 845.3977. General Procedure for Conjugate Synthesis. The peptide azide (2.5 μmol) and alkyne (7.5 μmol) were dissolved in 1:1 H2O/tBuOH (125 μL). A 100 mM solution of CuSO4 (15 μL, 1.5 μmol) and freshly prepared 500 mM solution of ascorbic acid (12 μL, 6.0 μmol) were added, and the mixture was stirred at 50 °C until complete consumption of the azide starting material observed by using MALDI. The product was then purified by HPLC.

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Author Contributions

S.A.C. performed all chemical synthesis and in vitro testing and wrote the manuscript. X.L., S.H., and M.Y. performed in vivo testing. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Angie Morales, Dr. Randy Whitall, Jing Zheng, and Bela Reiz for their assistance with mass spectrometry. These investigations were supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), Alberta Innovates Health Solutions, the Flight Attendant Medical Research Institute (FAMRI, Grant 103007), National Institutes of Health Grant AI109317-01A1, and NIH Grant AI101973-01 to M.W.

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ABBREVIATIONS USED ATCC, American Type Culture Collection; CFU, colony forming units; CuAAC, copper-catalyzed azide alkyne cycloaddition; DIPEA, diisopropylethylamine; DMF, dimethylformamide; DMSO, dimethylsulfoxide; Eryc, erythromycin A; Fmoc, fluorenylmethyloxycarbonyl; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; HBTU, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate; HPLC, high-performance liquid chromatography; LCMS, liquid chromatography−mass spectrometry; MALDI, matrix assisted laser desorption ionization; MDR, multidrug resistant; MIC, minimum inhibitory concentration; Oct, octanoyl; OM, outer-membrane; PEG, polyethylene glycol; Rif, rifampicin; SPPS, solid-phase peptide synthesis; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Tri, tridecaptin; Van, vancomycin; XDR, extremely drug resistant

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b01578.

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REFERENCES

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Synthetic procedures, compound characterization, and details on in vitro and in vivo testing (PDF) Molecular formula strings (CSV)

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DOI: 10.1021/acs.jmedchem.5b01578 J. Med. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jmedchem.5b01578 J. Med. Chem. XXXX, XXX, XXX−XXX